Corn Event MIR604

ABSTRACT

A novel transgenic corn event designated MIR604, is disclosed. The invention relates to DNA sequences of the recombinant constructs inserted into the corn genome and of genomic sequences flanking the insertion site that resulted in the MIR604 event. The invention further relates to assays for detecting the presence of the DNA sequences of MIR604, to corn plants and corn seeds comprising the genotype of MIR604 and to methods for producing a corn plant by crossing a corn plant comprising the MIR604 genotype with itself or another corn variety

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/556,260, filed on Mar. 25, 2004.

FIELD OF THE INVENTION

The present invention relates generally to the field of plant molecularbiology, plant transformation, and plant breeding. More specifically,the invention relates to insect resistant transgenic corn plantscomprising a novel transgenic genotype and to methods of detecting thepresence of the corn plant DNA in a sample and compositions thereof.

BACKGROUND

Plant pests are a major factor in the loss of the world's importantagricultural crops. About $8 billion are lost every year in the U.S.alone due to infestations of non-mammalian pests including insects.Species of corn rootworm are considered the most destructive corn pests.Important rootworm pest species include Diabrotica virgifera virgifera,the western corn rootworm; D. longicornis barberi, the northern cornrootworm, D. undecimpunctata howardi, the southern corn rootworm, and D.virgifera zeae, the Mexican corn rootworm.

Corn rootworm is mainly controlled by intensive applications of chemicalpesticides. Good corn rootworm control can thus be reached, but thesechemicals can sometimes also affect beneficial organisms. Anotherproblem resulting from the wide use of chemical pesticides is theappearance of resistant insect varieties. This has been partiallyalleviated by various resistance management practices, but there is anincreasing need for alternative pest control strategies. One suchalternative includes the expression of foreign genes encodinginsecticidal proteins in transgenic plants. This approach has providedan efficient means of protection against selected insect pests, andtransgenic plants expressing insecticidal toxins have beencommercialized, allowing farmers to reduce applications of chemicalinsecticides.

The expression of foreign genes in plants can to be influenced by theirchromosomal position, perhaps due to chromatin structure or theproximity of transcriptional regulation elements close to theintegration site (See for example, Weising et al., 1988, “Foreign Genesin Plants,” Ann. Rev. Genet. 22:421-477). Therefore, it is common toproduce hundreds of different events and screen those events for asingle event that has desired transgene expression levels and patternsfor commercial purposes. An event that has desired levels or patterns oftransgene expression is useful for introgressing the transgene intoother genetic backgrounds by sexual outcrossing using conventionalbreeding methods. Progeny of such crosses maintain the transgeneexpression characteristics of the original transformant. This strategyis used to ensure reliable gene expression in a number of varieties thatare well adapted to local growing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well-known nucleic acid detection methodincluding but not limited to thermal amplification (polymerase chainreaction (PCR)) using polynucleotide primers or DNA hybridization usingnucleic acid probes. Typically, for the sake of simplicity anduniformity of reagents and methodologies for use in detecting aparticular DNA construct that has been used for transforming variousplant varieties, these detection methods generally focus on frequentlyused genetic elements, for example, promoters, terminators, and markergenes, because for many DNA constructs, the coding sequence region isinterchangeable. As a result, such methods may not be useful fordiscriminating between constructs that differ only with reference to thecoding sequence. In addition, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted heterologous DNA (“flanking DNA”) is known.

The present invention includes an insect resistant transgenic corn eventthat has incorporated into its genome a cry3A055 gene, disclosed inInternational Publication No. WO 03/018810, published Mar. 6, 2003,which is herein incorporated by reference, encoding a Cry3A055insecticidal toxin, useful in controlling Diabrotica spp. insect pests.The transgenic corn event also has incorporated in its genome a pmigene, encoding a phosphomannose isomerase enzyme (PMI), disclosed inU.S. Pat. No. 5,767,378, which is herein incorporated by reference,useful as a selectable marker, which allows the plant to utilize mannoseas a carbon source. The present invention further includes novelisolated nucleic acid sequences which are unique to the transgenic cornevent, useful for identifying the transgenic corn event and fordetecting nucleic acids from the transgenic corn event in a biologicalsample, as well as kits comprising the reagents necessary for use indetecting these nucleic acids in a biological sample.

SUMMARY

The present invention is drawn to a transgenic corn event, designatedMIR604, comprising a novel transgenic genotype that comprises a cry3A055gene and a pmi gene which confers insect resistance and the ability toutilize mannose as a carbon source, respectively, to the MIR604 cornevent and progeny thereof. The invention also provides transgenic cornplants comprising the genotype of the invention, seed from transgeniccorn plants comprising the genotype of the invention, and to methods forproducing a transgenic corn plant comprising the genotype of theinvention by crossing a corn inbred comprising the genotype of theinvention with itself or another corn line of a different genotype. Thetransgenic corn plants of the invention may have essentially all of themorphological and physiological characteristics of the correspondingisogenic non-transgenic corn plant in addition to those conferred uponthe corn plant by the novel genotype of the invention. The presentinvention also provides compositions and methods for detecting thepresence of nucleic acids from event MIR604 based on the DNA sequence ofthe recombinant expression cassettes inserted into the corn genome thatresulted in the MIR604 event and of genomic sequences flanking theinsertion site. The MIR604 event can be further characterized byanalyzing expression levels of Cry3A055 and PMI proteins as well as bytesting efficacy against corn rootworm.

According to one aspect, the present invention provides an isolatednucleic acid molecule comprising at least 10 contiguous nucleotides of aheterologous DNA sequence inserted into the corn plant genome of cornevent MIR604 and at least 10 contiguous nucleotides of a corn plantgenome DNA flanking the point of insertion of a heterologous DNAsequence inserted into the corn plant genome of corn event MIR604. Theisolated nucleic acid molecule according to this aspect may comprise atleast 20 or at least 50 contiguous nucleotides of a heterologous DNAsequence inserted into the corn plant genome of corn event MIR604 and atleast 20 or at least 50 contiguous nucleotides of a corn plant genomeDNA flanking the point of insertion of a heterologous DNA sequenceinserted into the corn plant genome of corn event MIR604.

According to another aspect, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that comprises atleast one junction sequence of event MIR604 selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2, and complements thereof. Ajunction sequence spans the junction between the heterologous DNAcomprising the expression cassettes inserted into the corn genome andDNA from the corn genome flanking the insertion site and is diagnosticfor the MIR604 event.

According to another aspect, the present invention provides an isolatednucleic acid linking a heterologous DNA molecule to the corn plantgenome in corn event MIR604 comprising a sequence of from about 11 toabout 20 contiguous nucleotides selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, and complements thereof.

According to another aspect, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, and complements thereof.

According to another aspect of the invention, an amplicon comprising anucleic acid molecule of the invention is provided.

According to still another aspect of the invention, flanking sequenceprimers for detecting event MIR604 are provided. Such flanking sequenceprimers comprise an isolated nucleic acid sequence comprising at least10-15 contiguous nucleotides from nucleotides 1-801 as set forth in SEQID NO: 3 (arbitrarily designated herein as the 5′ flanking sequence), orthe complements thereof. In one embodiment of this aspect the flankingsequence primers are selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and complements thereof.

In another aspect of the invention, the flanking sequences primerscomprise an isolated nucleic acid sequence comprising at least 10-15contiguous nucleotides from nucleotides 507-1570 as set forth in SEQ IDNO: 4 (arbitrarily designated herein as the 3′ flanking sequence), orthe complements thereof. In one embodiment of this aspect the flankingsequence primers are selected from the group consisting of SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and complements thereof.

According to another aspect of the invention, primer pairs that areuseful for nucleic acid amplification, for example, are provided. Suchprimer pairs comprise a first primer comprising a nucleotide sequence ofat least 10-15 contiguous nucleotides in length which is or iscomplementary to one of the above-described genomic flanking sequences(SEQ ID NO: 3, or SEQ ID NO: 4) and a second primer comprising anucleotide sequence of at least 10-15 contiguous nucleotides ofheterologous DNA inserted into the event MIR604 genome. The secondprimer preferably comprises a nucleotide sequence which is or iscomplementary to the insert sequence adjacent to the plant genomicflanking DNA sequence as set forth in SEQ ID NO: 3 from nucleotideposition 802 through 1310 and in SEQ ID NO: 4 from nucleotide position 1through 506.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding to event MIR604 in a biological sample areprovided. Such methods comprise: (a) contacting the sample comprisingDNA with a pair of primers that, when used in a nucleic-acidamplification reaction with genomic DNA from corn event MIR604; producesan amplicon that is diagnostic for corn event MIR604; (b) performing anucleic acid amplification reaction, thereby producing the amplicon; and(c) detecting the amplicon. In one embodiment of this aspect, theamplicon comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:4, and complements thereof.

According to another aspect, the invention provides methods of detectingthe presence of a DNA corresponding to the MIR604 event in a biologicalsample. Such methods comprise: (a) contacting the sample comprising DNAwith a probe that hybridizes under high stringency conditions withgenomic DNA from corn event MIR604 and does not hybridize under highstringency conditions with DNA of a control corn plant; (b) subjectingthe sample and probe to high stringency hybridization conditions; and(c) detecting hybridization of the probe to the DNA.

According to another aspect of the invention, a kit is provided for thedetection of event MIR604 nucleic acids in a biological sample. The kitincludes at least one DNA sequence comprising a sufficient length ofpolynucleotides which is or is complementary to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO: 4, wherein the DNA sequences are usefulas primers or probes that hybridize to isolated DNA from event MIR604,and which, upon amplification of or hybridization to a nucleic acidsequence in a sample followed by detection of the amplicon orhybridization to the target sequence, are diagnostic for the presence ofnucleic acid sequences from event MIR604 in the sample. The kit furtherincludes other materials necessary to enable nucleic acid hybridizationor amplification methods.

In another aspect, the present invention provides a method of detectingcorn event MIR604 protein in a biological sample comprising: (a)extracting protein from a sample of corn event MIR604 tissue; (b)assaying the extracted protein using an immunological method comprisingantibody specific for the insecticidal or selectable marker proteinproduced by the MIR604 event; and (c) detecting the binding of saidantibody to the insecticidal or selectable marker protein.

In another aspect, the present invention provides a biological samplederived from a event MIR604 corn plant, tissue, or seed, wherein thesample comprises a nucleotide sequence which is or is complementary to asequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 2, and wherein the sequence is detectable in the sample using anucleic acid amplification or nucleic acid hybridization method. In oneembodiment of this aspect, the sample is selected from the groupconsisting of corn flour, corn meal, corn syrup, corn oil, cornstarch,and cereals manufactured in whole or in part to contain cornby-products.

In another aspect, the present invention provides an extract derivedfrom a event MIR604 corn plant, tissue, or seed comprising a nucleotidesequence which is or is complementary to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In oneembodiment of this aspect, the sequence is detectable in the extractusing a nucleic acid amplification or nucleic acid hybridization method.In another embodiment of this aspect, the sample is selected from thegroup consisting of corn flour, corn meal, corn syrup, corn oil,cornstarch, and cereals manufactured in whole or in part to contain cornby-products.

According to another aspect of the invention, corn plants and seedscomprising the nucleic acid molecules of the invention are provided.

According to another aspect, the present invention provides a method forproducing a corn plant resistant to at least corn rootworm infestationcomprising: (a) sexually crossing a first parent corn plant with asecond parent corn plant, wherein said first or second parent corn plantcomprises corn event MIR604 DNA, thereby producing a plurality of firstgeneration progeny plants; (b) selecting a first generation progenyplant that is resistant to at least corn rootworm infestation; (c)selfing the first generation progeny plant, thereby producing aplurality of second generation progeny plants; (d) selecting from thesecond generation progeny plants, a plant that is at least resistant tocorn rootworm infestation; wherein the second generation progeny plantscomprise a nucleotide sequence selected from the group consisting of SEQID NO: 1 and SEQ ID NO: 2.

According to yet another aspect, the present invention provides a methodfor producing corn seed comprising crossing a first parent corn plantwith a second parent corn plant and harvesting the resultant firstgeneration corn seed, wherein the first or second parent corn plant isan inbred corn plant of the invention.

According to another aspect, the present invention provides a method ofproducing hybrid corn seeds comprising the steps of: (a) planting seedsof a first inbred corn line according to the invention and seeds of asecond inbred corn line having a different genotype; (b) cultivatingcorn plants resulting from said planting until time of flowering; (c)emasculating flowers of corn plants of one of the corn inbred lines; (d)allowing pollination of the other inbred line to occur, and (e)harvesting the hybrid seed produced thereby.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the 5′ genome-insert junction.SEQ ID NO: 2 is the 3′ insert-genome junction.SEQ ID NO: 3 is the 5′ genome+insert sequence.SEQ ID NO: 4 is the 3′ insert+genome sequence.SEQ ID NO: 5 is corn genome flanking 5′ to insert.SEQ ID NO: 6 is corn genome flanking 3′ to insert.SEQ ID Nos: 7-15 are 5′ flanking sequence primers useful in the presentinvention.SEQ ID Nos: 16-20 are MTL promoter sequence primers useful in thepresent invention.SEQ ID Nos: 21-28 are cry3A055 sequence primers useful in the presentinvention.SEQ ID Nos: 29-30 are ZmUbiInt sequence primers useful in the presentinvention.SEQ ID Nos: 31-37 are pmi sequence primers useful in the presentinvention.SEQ ID NO: 38 is a NOS sequence primer useful in the present invention.SEQ ID NO: 39-46 are 3′ flanking sequence primers useful in the presentinvention.SEQ ID Nos: 47-49 are cry3A055 TAQMAN primers and probe.SEQ ID Nos: 50-52 are pmi TAQMAN primers and probe.SEQ ID NO: 53-55 are ZmADH TAQMAN primers and probe.SEQ ID NO: 56 is a MIR604 probe useful in the present invention.SEQ ID NO: 57 is the sequence for the right border region.SEQ ID NO: 58 is the sequence of the MTL promoter.SEQ ID NO: 59 is the sequence of the cry3A055 gene.SEQ ID NO: 60 is the sequence of the NOS terminator.SEQ ID NO: 61 is the sequence of the ZmUbInt promoter.SEQ ID NO: 62 is the sequence of the pmi gene.SEQ ID NO: 63 is the sequence of the left border region.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a plant expression vector designated pZM26. Mapidentifies KpnI restriction site used for Southern analysis.

FIG. 2 is a graphical map illustrating the organization of the elementscomprising the heterologous nucleic acid sequences inserted into thecorn event MIR604 genome and sets forth the relative positions at whichthe inserted nucleic acid sequences are linked to corn genomic DNAsequences which flank the ends of the inserted heterologous DNAsequences. 1=5′flanking plant genome (SEQ ID NO: 5); 2=right borderregion (SEQ ID NO: 57); 3=MTL promoter (SEQ ID NO: 58); 4=cry3A055 gene(SEQ ID NO: 59); 5=NOS terminator (SEQ ID NO: 60); 6=ZmUbINT promoter(SEQ ID NO: 61); 7=pmi gene (SEQ ID NO: 62); 8=NOS terminator (SEQ IDNO: 60); 9=left border region (SEQ ID NO: 63); and 10=3′ flanking plantgenome (SEQ ID NO: 6).

DEFINITIONS

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms usedherein are to be understood according to conventional usage by those ofordinary skill in the relevant art. Definitions of common terms inmolecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5^(th) edition, Springer-Verlag: NewYork, 1994.

As used herein, the term “amplified” means the construction of multiplecopies of a nucleic acid molecule or multiple copies complementary tothe nucleic acid molecule using at least one of the nucleic acidmolecules as a template. Amplification systems include the polymerasechain reaction (PCR) system, ligase chain reaction (LCR) system, nucleicacid sequence based amplification (NASBA, Cangene, Mississauga,Ontario), Q-Beta Replicase systems, transcription-based amplificationsystem (TAS), and strand displacement amplification (SDA). See, e.g.,Diagnostic Molecular Microbiology: Principles and Applications, D. H.Persing et al., Ed., American Society for Microbiology, Washington, D.C.(1993). The product of amplification is termed an amplicon.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

“Detection kit” as used herein refers to a kit used to detect thepresence or absence of DNA from MIR604 plants in a sample comprisingnucleic acid probes and primers of the present invention, whichhybridize specifically under high stringency conditions to a target DNAsequence, and other materials necessary to enable nucleic acidhybridization or amplification methods.

As used herein the term transgenic “event” refers to a recombinant plantproduced by transformation and regeneration of a single plant cell withheterologous DNA, for example, an expression cassette that includes agene of interest. The term “event” refers to the original transformantand/or progeny of the transformant that include the heterologous DNA.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another corn line. Even after repeatedbackcrossing to a recurrent parent, the inserted DNA and the flankingDNA from the transformed parent is present in the progeny of the crossat the same chromosomal location. Normally, transformation of planttissue produces multiple events, each of which represent insertion of aDNA construct into a different location in the genome of a plant cell.Based on the expression of the transgene or other desirablecharacteristics, a particular event is selected. Thus, “event MIR604”,“MIR604” or “MIR604 event” as used herein, means the original MIR604transformant and/or progeny of the MIR604 transformant.

“Expression cassette” as used herein means a nucleic acid moleculecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassette may alsocomprise sequences not necessary in the direct expression of anucleotide sequence of interest but which are present due to convenientrestriction sites for removal of the cassette from an expression vector.The expression cassette comprising the nucleotide sequence of interestmay be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular nucleic acid sequence of theexpression cassette does not occur naturally in the host cell and musthave been introduced into the host cell or an ancestor of the host cellby a transformation process known in the art. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter that initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, such as aplant, the promoter can also be specific to a particular tissue, ororgan, or stage of development. An expression cassette, or fragmentthereof, can also be referred to as “inserted sequence” or “insertionsequence” when transformed into a plant.

A “gene” is a defined region that is located within a genome and that,besides the aforementioned coding nucleic acid sequence, comprisesother, primarily regulatory, nucleic acid sequences responsible for thecontrol of the expression, that is to say the transcription andtranslation, of the coding portion. A gene may also comprise other 5′and 3′ untranslated sequences and termination sequences. Furtherelements that may be present are, for example, introns.

“Gene of interest” refers to any gene which, when transferred to aplant, confers upon the plant a desired characteristic such asantibiotic resistance, virus resistance, insect resistance, diseaseresistance, or resistance to other pests, herbicide tolerance, improvednutritional value, improved performance in an industrial process oraltered reproductive capability. The “gene of interest” may also be onethat is transferred to plants for the production of commerciallyvaluable enzymes or metabolites in the plant.

“Genotype” as used herein is the genetic material inherited from parentcorn plants not all of which is necessarily expressed in the descendantcorn plants. The MIR604 genotype refers to the heterologous geneticmaterial transformed into the genome of a plant as well as the geneticmaterial flanking the inserted sequence.

A “heterologous” nucleic acid sequence is a nucleic acid sequence notnaturally associated with a host cell into which it is introduced,including non-naturally occurring multiple copies of a naturallyoccurring nucleic acid sequence.

A “homologous” nucleic acid sequence is a nucleic acid sequencenaturally associated with a host cell into which it is introduced.

“Operably-linked” refers to the association of nucleic acid sequences ona single nucleic acid fragment so that the function of one affects thefunction of the other. For example, a promoter is operably-linked with acoding sequence or functional RNA when it is capable of affecting theexpression of that coding sequence or functional RNA (i.e., that thecoding sequence or functional RNA is under the transcriptional controlof the promoter). Coding sequences in sense or antisense orientation canbe operably-linked to regulatory sequences.

“Primers” as used herein are isolated nucleic acids that are annealed toa complimentary target DNA strand by nucleic acid hybridization to forma hybrid between the primer and the target DNA strand, then extendedalong the target DNA strand by a polymerase, such as DNA polymerase.Primer pairs or sets can be used for amplification of a nucleic acidmolecule, for example, by the polymerase chain reaction (PCR) or otherconventional nucleic-acid amplification methods.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme. Such aprobe is complimentary to a strand of a target nucleic acid, in the caseof the present invention, to a strand of genomic DNA from corn event,MIR604. The genomic DNA of MIR604 can be from a corn plant or from asample that includes DNA from the event. Probes according to the presentinvention include not only deoxyribonucleic or ribonucleic acids butalso polyamides and other probe materials that bind specifically to atarget DNA sequence and can be used to detect the presence of thattarget DNA sequence.

Primers and probes are generally between 10 and 15 nucleotides or morein length, Primers and probes can also be at least 20 nucleotides ormore in length, or at least 25 nucleotides or more, or at least 30nucleotides or more in length. Such primers and probes hybridizespecifically to a target sequence under high stringency hybridizationconditions. Primers and probes according to the present invention mayhave complete sequence complementarity with the target sequence,although probes differing from the target sequence and which retain theability to hybridize to target sequences may be designed by conventionalmethods.

“Stringent conditions” or “stringent hybridization conditions” includereference to conditions under which a probe will hybridize to its targetsequence, to a detectably greater degree than to other sequences.Stringent conditions are target-sequence-dependent and will differdepending on the structure of the polynucleotide. By controlling thestringency of the hybridization and/or wash conditions, target sequencescan be identified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Longer sequences hybridize specificallyat higher temperatures. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, Part I, Chapter 2 “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier: New York; andCurrent Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds.,Greene Publishing and Wiley-Interscience: New York (1995), and alsoSambrook et al. (2001) Molecular Cloning: A Laboratory Manual (5^(th)Ed. Cols Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. Generally, high stringency hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH. The T_(m) is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. Typically, under high stringency conditions a probe willhybridize to its target subsequence, but to no other sequences.

An example of high stringency hybridization conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of very high stringency wash conditions is 0.15MNaCl at 72° C. for about 15 minutes. An example of high stringency washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook,infra, for a description of SSC buffer).

Exemplary hybridization conditions for the present invention includehybridization in 7% SDS, 0.25 M NaPO₄ pH 7.2 at 67° C. overnight,followed by two washings in 5% SDS, 0.20 M NaPO₄ pH7.2 at 65° C. for 30minutes each wash, and two washings in 1% SDS, 0.20 M NaPO₄ pH7.2 at 65°C. for 30 minutes each wash. An exemplary medium stringency wash for aduplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15minutes. An exemplary low stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes.

For probes of about 10 to 50 nucleotides, high stringency conditionstypically involve salt concentrations of less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.High stringency conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other underhigh stringency conditions are still substantially identical if theproteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

The following are exemplary sets of hybridization/wash conditions thatmay be used to hybridize nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the present invention: areference nucleotide sequence preferably hybridizes to the referencenucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., moredesirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably stillin 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C. The sequences of the present invention may bedetected using all the above conditions. For the purposes of definingthe invention, the high stringency conditions are used.

“Transformation” is a process for introducing heterologous nucleic acidinto a host cell or organism. In particular, “transformation” means thestable integration of a DNA molecule into the genome of an organism ofinterest.

“Transformed/transgenic/recombinant” refer to a host organism such as abacterium or a plant into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome of the host or the nucleic acid molecule can also be presentas an extrachromosomal molecule. Such an extrachromosomal molecule canbe auto-replicating. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof. A “non-transformed”,“non-transgenic”, or “non-recombinant” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule. As used herein, “transgenic” refersto a plant, plant cell, or multitude of structured or unstructured plantcells having integrated, via well known techniques of geneticmanipulation and gene insertion, a sequence of nucleic acid representinga gene of interest into the plant genome, and typically into achromosome of a cell nucleus, mitochondria or other organelle containingchromosomes, at a locus different to, or in a number of copies greaterthan, that normally present in the native plant or plant cell.Transgenic plants result from the manipulation and insertion of suchnucleic acid sequences, as opposed to naturally occurring mutations, toproduce a non-naturally occurring plant or a plant with a non-naturallyoccurring genotype. Techniques for transformation of plants and plantcells are well known in the art and may comprise for exampleelectroporation, microinjection, Agrobacterium-mediated transformation,and ballistic transformation.

The nomenclature for DNA bases and amino acids as set forth in 37 C.F.R§ 1.822 is used herein.

DETAILED DESCRIPTION

This invention relates to a genetically improved line of corn thatproduces the insect control protein, Cry3A055, and a phosphomannoseisomerase enzyme (PMI) that allows the plant to utilize mannose as acarbon source. The invention is particularly drawn to a transgenic cornevent designated MIR604 comprising a novel genotype, as well as tocompositions and methods for detecting nucleic acids from this event ina biological sample. The invention is further drawn to corn plantscomprising the MIR604 genotype, to transgenic seed from the corn plants,and to methods for producing a corn plant comprising the MIR604 genotypeby crossing a corn inbred comprising the MIR604 genotype with itself oranother corn line. Corn plants comprising the MIR604 genotype of theinvention are useful in controlling coleopteran insect pests includingDiabrotica virgira virgifera, the western corn rootworm, D. virgiferazeae, the Mexican corn rootworm, and D. lonigicornis barberi, thenorthern corn rootworm. Corn plants comprising the MIR604 genotype ofthe invention are also able to utilize mannose as a carbon source.

In one embodiment, the present invention encompasses an isolated nucleicacid molecule comprising at least 10 or more (for example 15, 20, 25, or50) contiguous nucleotides of a heterologous DNA sequence inserted intothe corn plant genome of corn event MIR604 and at least 10 or more (forexample 15, 20, 25, or 50) contiguous nucleotides of a corn plant genomeDNA flanking the point of insertion of a heterologous DNA sequenceinserted into the corn plant genome of corn event MIR604. Also includedare nucleotide sequences that comprise 10 or more nucleotides ofcontiguous insert sequence from event MIR604 and at lease one nucleotideof flanking DNA from event MIR604 adjacent to the insert sequence. Suchnucleotide sequences are diagnostic for event MIR604. Nucleic acidamplification of genomic DNA from the MIR604 event produces an ampliconcomprising such diagnostic nucleotide sequences.

In another embodiment, the invention encompasses an isolated nucleicacid molecule comprising a nucleotide sequence which comprises at leastone junction sequence of event MIR604 selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, and complements thereof, wherein ajunction sequence spans the junction between a heterologous expressioncassette inserted into the corn genome and DNA from the corn genomeflanking the insertion site and is diagnostic for the event.

In another embodiment, the present invention encompasses an isolatednucleic acid linking a heterologous DNA molecule to the corn plantgenome in corn event MIR604 comprising a sequence of from about 11 toabout 20 contiguous nucleotides selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, and the complements thereof.

In another embodiment, the invention encompasses an isolated nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,and complements thereof.

In one embodiment of the present invention, an amplicon comprising anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof isprovided.

In another embodiment, the present invention encompasses flankingsequence primers for detecting event MIR604. Such flanking sequenceprimers comprise an isolated nucleic acid sequence comprising at least10-15 contiguous nucleotides from nucleotides 1-801 of SEQ ID NO: 3(arbitrarily designated herein as the 5′ flanking sequence), or thecomplements thereof. In one aspect of this embodiment the flankingsequence primers are selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and complements thereof.

In another embodiment, the present invention encompasses flankingsequence primers that comprise at least 10-15 contiguous nucleotidesfrom nucleotides 507-1570 of SEQ ID NO: 4 (arbitrarily designated hereinas the 3′ flanking sequence), or the complements thereof. In one aspectof this embodiment the flanking sequence primers are selected from thegroup consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ IDNO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, andcomplements thereof.

In still another embodiment, the present invention encompasses a pair ofpolynucleotide primers comprising a first polynucleotide primer and asecond polynucleotide primer which function together in the presence ofa corn event MIR604 DNA template in a sample to produce an amplicondiagnostic for the corn event MIR604, wherein the first primer sequenceis or is complementary to a corn plant genome flanking the point ofinsertion of a heterologous DNA sequence inserted into the corn plantgenome of corn event MIR604, and the second polynucleotide primersequence is or is complementary to the heterologous DNA sequenceinserted into the corn plant genome of the corn event MIR604.

In one aspect of this embodiment the first polynucleotide primercomprises at least 10 contiguous nucleotides from position 1-801 of SEQID NO: 3 or complements thereof. In a further aspect of this embodiment,the first polynucleotide primer comprises the nucleotide sequence setforth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, orthe complements thereof. In another aspect of this embodiment the firstpolynucleotide primer least 10 contiguous nucleotides from position507-1570 of SEQ ID NO: 4 or complements thereof. In another aspect ofthis embodiment, the first polynucleotide primer comprises thenucleotide sequence set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ IDNO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQID NO: 46, or the complements thereof. In yet another aspect of thisembodiment, the second polynucleotide primer comprises at least 10contiguous nucleotides of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or thecomplements thereof. In still a further aspect of this embodiment, thesecond polynucleotide primer comprises the nucleotide sequence set forthin SEQ ID NO: 16 to SEQ ID NO: 38, or the complements thereof.

In another aspect of this embodiment, the first polynucleotide primer,which is set forth in SEQ ID NO: 15, and the second polynucleotideprimer which is set forth in SEQ ID NO: 28, function together in thepresence of a corn event MIR604 DNA template in a sample to produce anamplicon diagnostic for the corn event MIR604 as described in Example 4.In another aspect of this embodiment, the first polynucleotide primer,which is set forth in SEQ ID NO: 45, and the second polynucleotideprimer which is set forth in SEQ ID NO: 27, function together in thepresence of a corn event MIR604 DNA template in a sample to produce anamplicon diagnostic for the corn event MIR604 as described in Example 4.

Of course, it is well within the skill in the art to obtain additionalsequence further out into the genome sequence flanking either end of theinserted heterologous DNA sequences for use as a primer sequence thatcan be used in such primer pairs for amplifying the sequences that arediagnostic for the MIR604 event. For the purposes of this disclosure,the phrase “further out into the genome sequence flanking either end ofthe inserted heterologous DNA sequences” refers specifically to asequential movement away from the ends of the inserted heterologous DNAsequences, the points at which the inserted DNA sequences are adjacentto native genomic DNA sequence, and out into the genomic DNA of theparticular chromosome into which the heterologous DNA sequences wereinserted. Preferably, a primer sequence corresponding to orcomplementary to a part of the insert sequence should prime thetranscriptional extension of a nascent strand of DNA or RNA toward thenearest flanking sequence junction. Consequently, a primer sequencecorresponding to or complementary to a part of the genomic flankingsequence should prime the transcriptional extension of a nascent strandof DNA or RNA toward the nearest flanking sequence junction. A primersequence can be, or can be complementary to, a heterologous DNA sequenceinserted into the chromosome of the plant, or a genomic flankingsequence. One skilled in the art would readily recognize the benefit ofwhether a primer sequence would need to be, or would need to becomplementary to, the sequence as set forth within the insertedheterologous DNA sequence or as set forth in SEQ ID NO: 3 or SEQ ID NO:4 depending upon the nature of the product desired to be obtainedthrough the use of the nested set of primers intended for use inamplifying a particular flanking sequence containing the junctionbetween the genomic DNA sequence and the inserted heterologous DNAsequence.

In another embodiment, the present invention encompasses a method ofdetecting the presence of DNA corresponding to the event MIR604 in abiological sample, wherein the method comprises: (a) contacting thesample comprising DNA with a probe that hybridizes under high stringencyconditions with genomic DNA from corn event MIR604 and does nothybridize under high stringency conditions with DNA of a control cornplant; (b) subjecting the sample and probe to high stringencyhybridization conditions; and (c) detecting hybridization of the probeto the DNA. In one aspect of this embodiment the amplicon comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and complements thereof.

In another embodiment, the present invention encompasses a method ofdetecting the presence of a DNA corresponding to the MIR604 event in abiological sample, wherein the method comprises: (a) contacting thesample comprising DNA with a probe that hybridizes under high stringencyconditions with genomic DNA from corn event MIR604 and does nothybridize under high stringency conditions with DNA of a control cornplant; (b) subjecting the sample and probe to high stringencyhybridization conditions; and (c) detecting hybridization of the probeto the DNA. Detection can be by any means well known in the artincluding but not limited to fluorescent, chemiluminescent,radiological, immunological, or otherwise. In the case in whichhybridization is intended to be used as a means for amplification of aparticular sequence to produce an amplicon which is diagnostic for theMIR604 corn event, the production and detection by any means well knownin the art of the amplicon is intended to be indicative of the intendedhybridization to the target sequence where one probe or primer isutilized, or sequences where two or more probes or primers are utilized.The term “biological sample” is intended to comprise a sample thatcontains or is suspected of containing a nucleic acid comprising frombetween five and ten nucleotides either side of the point at which oneor the other of the two terminal ends of the inserted heterologous DNAsequence contacts the genomic DNA sequence within the chromosome intowhich the heterologous DNA sequence was inserted, herein also known asthe junction sequences. In addition, the junction sequence comprises aslittle as two nucleotides: those being the first nucleotide within theflanking genomic DNA adjacent to and covalently linked to the firstnucleotide within the inserted heterologous DNA sequence.

In yet another embodiment, the present invention encompasses a kit fordetecting the presence of MIR604 nucleic acids in a biological sample,wherein the kit comprises at least one nucleic acid molecule ofsufficient length of contiguous nucleotides homologous or complementaryto a nucleotide sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, that functions as aDNA primer or probe specific for event MIR604, and other materialsnecessary to enable nucleic acid hybridization or amplification. Avariety of detection methods can be used including TAQMAN (PerkinElmer), thermal amplification, ligase chain reaction, southernhybridization, ELISA methods, and calorimetric and fluorescent detectionmethods. In particular the present invention provides for kits fordetecting the presence of the target sequence, i.e., at least one of thejunctions of the insert DNA with the genomic DNA of the corn plant inMIR604, in a sample containing genomic nucleic acid from MIR604. The kitis comprised of at least one polynucleotide capable of binding to thetarget site or substantially adjacent to the target site and at leastone means for detecting the binding of the polynucleotide to the targetsite. The detecting means can be fluorescent, chemiluminescent,calorimetric, or isotopic and can be coupled at least with immunologicalmethods for detecting the binding. A kit is also envisioned which candetect the presence of the target site in a sample, i.e., at least oneof the junctions of the insert DNA with the genomic DNA of the cornplant in MIR604, taking advantage of two or more polynucleotidesequences which together are capable of binding to nucleotide sequencesadjacent to or within about 100 base pairs, or within about 200 basepairs, or within about 500 base pairs or within about 1000 base pairs ofthe target sequence and which can be extended toward each other to forman amplicon which contains at least the target site

In another embodiment, the present invention encompasses a method fordetecting event MIR604 protein in a biological sample, the methodcomprising: (a) extracting protein from a sample of corn event MIR604tissue; (b) assaying the extracted protein using an immunological methodcomprising antibody specific for the insecticidal or selectable markerprotein produced by the MIR604 event; and (c) detecting the binding ofsaid antibody to the insecticidal or selectable marker protein.

Another embodiment of the present invention encompasses a corn plant, orparts thereof, comprising the genotype of the transgenic event MIR604,wherein the genotype comprises the nucleotide sequence set forth in SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or the complementsthereof. In one aspect of this embodiment, the corn plant is from theinbred corn lines CG5NA58, CG5NA58A, CG3ND97, CG5NA01, CG5NF22, CG4NU15,CG00685, CG00526, CG00716, NP904, NP948, NP934, NP982, NP991, NP993,NP2010, NP2013, NP2015, NP2017, NP2029, NP2031, NP2034, NP2045, NP2052,NP2138, NP2151, NP2166, NP2161, NP2171, NP2174, NP2208, NP2213, NP2222,NP2275, NP2276, NP2316, BCTT609, AF031, H8431, 894, BUTT201, R327H,2044BT, and 2070BT. One skilled in the art will recognize however, thatthe MIR604 genotype can be introgressed into any plant variety that canbe bred with corn, including wild maize species, and thus the preferredinbred lines of this embodiment are not meant to be limiting.

In another embodiment, the present invention encompasses a corn plantcomprising at least a first and a second DNA sequence linked together toform a contiguous nucleotide sequence, wherein the first DNA sequence iswithin a junction sequence and comprises at least about II contiguousnucleotides selected from the group consisting of nucleotides 792-811 ofSEQ ID NO: 3; nucleotides 497-516 of SEQ ID NO: 4; SEQ ID NO: 5; SEQ IDNO: 6; and complements thereof, wherein the second DNA sequence iswithin the heterologous insert DNA sequence selected from the groupconsisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and complementsthereof; and wherein the first and the second DNA sequences are usefulas nucleotide primers or probes for detecting the presence of corn eventMIR604 nucleic acid sequences in a biological sample. In one aspect ofthis embodiment, the nucleotide primers are used in a DNA amplificationmethod to amplify a target DNA sequence from template DNA extracted fromthe corn plant and the corn plant is identifiable from other corn plantsby the production of an amplicon corresponding to a DNA sequencecomprising SEQ ID NO: 1 or SEQ ID NO: 2

Corn plants of the invention can be further characterized in thatdigesting the plant's genomic DNA with the restriction endonuclease KpnIresults in a single cry3A055 hybridizing band using a cry3A055-specificprobe under high stringency conditions. Exemplified herein is a cry3A055probe comprising a nucleotide sequence set forth in SEQ ID NO: 56 or SEQID 59.

Corn plants of the invention can be further characterized in thatdigesting the plant's genomic DNA with the restriction endonuclease KpnIresults in a single pmi hybridizing band using a pmi-specific probeunder high stringency conditions. Exemplified herein is a pmi probecomprising a nucleotide sequence set forth in SEQ ID NO: 62.

In one embodiment, the present invention provides a corn plant, whereinthe MIR604 genotype confers upon the corn plant resistance to insects orthe ability to utilize mannose. In one aspect of this embodiment, thegenotype conferring resistance to insects upon the corn plant comprisesa cry3A055 gene. In another aspect of this embodiment, the genotypeconferring upon the corn plant the ability to utilize mannose comprisesa pmi gene.

In one embodiment, the present invention provides a biological samplederived from a event MIR604 corn plant, tissue, or seed, wherein thesample comprises a nucleotide sequence which is or is complementary to asequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 2, and wherein the sequence is detectable in the sample using anucleic acid amplification or nucleic acid hybridization method. In oneaspect of this embodiment, the sample is selected from corn flour, cornsyrup, corn oil, corn starch, and cereals manufactured in whole or inpart to contain corn products.

In another embodiment, the present invention provides an extract derivedfrom a event MIR604 corn plant, tissue, or seed comprising a nucleotidesequence which is or is complementary to a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In oneaspect of this embodiment, the sequence is detected in the extract usinga nucleic acid amplification or nucleic acid hybridization method. Inanother aspect of this embodiment, the sample is selected from cornflour, corn syrup, corn oil, cornstarch, and cereals manufactured inwhole or in part to contain corn products.

In yet another embodiment, the present invention provides a method forproducing a corn plant resistant to at least corn rootworm infestationcomprising: (a) sexually crossing a first parent corn plant with asecond parent corn plant, wherein said first or second parent corn plantcomprises corn event MIR604 DNA, thereby producing a plurality of firstgeneration progeny plants; (b) selecting a first generation progenyplant that is resistant to at least corn rootworm infestation; (c)selfing the first generation progeny plant, thereby producing aplurality of second generation progeny plants; and (d) selecting fromthe second generation progeny plants, a plant that is at least resistantto corn rootworm infestation; wherein the second generation progenyplants comprise a nucleotide sequence selected from the group consistingof SEQ ID NO: 1 and SEQ ID NO: 2.

In another embodiment, the present invention provides a method ofproducing hybrid corn seeds comprising: (a) planting seeds of a firstinbred corn line comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ IDNO: 4, and seeds of a second inbred line having a different genotype;(b) cultivating corn plants resulting from said planting until time offlowering; (c) emasculating said flowers of plants of one of the corninbred lines; (d) sexually crossing the two different inbred lines witheach other; and (e) harvesting the hybrid seed produced thereby. In oneaspect of this embodiment, the first inbred corn line provides thefemale parents. In another aspect of this embodiment, the first inbredcorn line provides the male parents. The present invention alsoencompasses the hybrid seed produced by the embodied method and hybridplants grown from the seed.

One skilled in the art will recognize that the transgenic genotype ofthe present invention can be introgressed by breeding into other cornlines comprising different transgenic genotypes. For example, a corninbred comprising the transgenic genotype of the present invention canbe crossed with a corn inbred comprising the transgenic genotype of thelepidopteran resistant Bt11 event, which is known in the art, thusproducing corn seed that comprises both the transgenic genotype of theinvention and the Bt11 transgenic genotype. Examples of other transgenicevents which can be crossed with an inbred of the present inventioninclude, the glyphosate tolerant events GA21 and NK603, the glyphosatetolerant/lepidopteran insect resistant MON802 event, the lepidopteranresistant DBT418 event, the lepidopteran resistant event DAS-06275-8,the male sterile event MS3, the phosphinothricin tolerant event B16, thelepidopteran insect resistant event MON 80100, the phosphinothricintolerant events T14 and T25, the lepidopteran insect resistant event176, and the coleopteran resistant event MON863, all of which are knownin the art. It will be further recognized that other combinations can bemade with the transgenic genotype of the invention and thus theseexamples should not be viewed as limiting.

One skilled in the art will also recognize that transgenic corn seedcomprising the transgenic genotype of the present invention can betreated with various seed-treatment chemicals, including insecticides,to augment or syngergize the insecticidal activity of the Cry3A055protein. For example, the transgenic corn seed of the present inventioncan be treated with the commercial insecticide Cruiser®. Such acombination may used to increase the spectrum of activity and toincrease the efficacy of the expressed protein and chemical.

Breeding

The transgenic genotype of the present invention can be introgressed inany corn inbred or hybrid using art recognized breeding techniques. Thegoal of plant breeding is to combine in a single variety or hybridvarious desirable traits. For field crops, these traits may includeresistance to insects and diseases, tolerance to herbicides, toleranceto heat and drought, reducing the time to crop maturity, greater yield,and better agronomic quality. With mechanical harvesting of many crops,uniformity of plant characteristics such as germination and standestablishment, growth rate, maturity, and plant and ear height, isimportant.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Corn can be bred by both self-pollination and cross-pollinationtechniques. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ears.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of corn hybrids, which relies upon some sort of malesterility system. There are several options for controlling malefertility available to breeders, such as: manual or mechanicalemasculation (or detasseling), cytoplasmic male sterility, genetic malesterility, gametocides and the like.

Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using one of many methods of conferring genetic malesterility in the art, each with its own benefits and drawbacks. Thesemethods use a variety of approaches such as delivering into the plant agene encoding a cytotoxic substance associated with a male tissuespecific promoter or an antisense system in which a gene critical tofertility is identified and an antisense to that gene is inserted in theplant (see: Fabinjanski, et al. EPO 89/3010153.8 publication no. 329,308and PCT application PCT/CA90/00037 published as WO 90/08828).

Development of Corn Inbred Lines

The use of male sterile inbreds is but one factor in the production ofcorn hybrids. Plant breeding techniques known in the art and used in acorn plant breeding program include, but are not limited to, recurrentselection, backcrossing, pedigree breeding, restriction lengthpolymorphism enhanced selection, genetic marker enhanced selection andtransformation. The development of corn hybrids in a corn plant breedingprogram requires, in general, the development of homozygous inbredlines, the crossing of these lines, and the evaluation of the crosses.Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Corn plant breedingprograms combine the genetic backgrounds from two or more inbred linesor various other germplasm sources into breeding pools from which newinbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which of thosehave commercial potential. Plant breeding and hybrid development, aspracticed in a corn plant-breeding program, are expensive andtime-consuming processes.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F₁→F₂; F₂→F₃; F₃→F₄; F₄→F.₅; etc.

Recurrent selection breeding, backcrossing for example, can be used toimprove an inbred line and a hybrid that is made using those inbreds.Backcrossing can be used to transfer a specific desirable trait from oneinbred or source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred(recurrent parent) to a donor inbred (non-recurrent parent), thatcarries the appropriate gene(s) for the trait in question. The progenyof this cross is then mated back to the superior recurrent parentfollowed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait, the progenywill be homozygous for loci controlling the characteristic beingtransferred, but will be like the superior parent for essentially allother genes. The last backcross generation is then selfed to give purebreeding progeny for the gene(s) being transferred. A hybrid developedfrom inbreds containing the transferred gene(s) is essentially the sameas a hybrid developed from the same inbreds without the transferredgene(s).

Elite inbred lines, that is, pure breeding, homozygous inbred lines, canalso be used as starting materials for breeding or source populationsfrom which to develop other inbred lines. These inbred lines derivedfrom elite inbred lines can be developed using the pedigree breeding andrecurrent selection breeding methods described earlier. As an example,when backcross breeding is used to create these derived lines in a cornplant-breeding program, elite inbreds can be used as a parental line orstarting material or source population and can serve as either the donoror recurrent parent.

Development of Corn Hybrids

A single cross corn hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F₁. In thedevelopment of commercial hybrids in a corn plant-breeding program, onlythe F₁ hybrid plants are sought. Preferred F₁ hybrids are more vigorousthan their inbred parents. This hybrid vigor, or heterosis, can bemanifested in many polygenic traits, including increased vegetativegrowth and increased yield.

The development of a corn hybrid in a corn plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, although different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrid progeny (F₁). Duringthe inbreeding process in corn, the vigor of the lines decreases. Vigoris restored when two different inbred lines are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid between a definedpair of inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained. Much of the hybrid vigor exhibited by F₁ hybrids is lost inthe next generation (F₂). Consequently, seed from hybrids is not usedfor planting stock.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for self-pollination. This inadvertentlyself-pollinated seed may be unintentionally harvested and packaged withhybrid seed.

Once the seed is planted, it is possible to identify and select theseself-pollinated plants. These self-pollinated plants will be geneticallyequivalent to the female inbred line used to produce the hybrid.

As is readily apparent to one skilled in the art, the foregoing are onlysome of the various ways by which the inbred of the present inventioncan be obtained by those looking to introgress the transgenic genotypeof the invention into other corn lines. Other means are available, andthe above examples are illustrative only.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Ausubel (ed.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc.(1994); J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3dEd., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press(2001); and by T. J. Silhavy, M. L. Berman, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984).

Example 1 Transformation and Selection of the MIR604 Event

The MIR604 event was produced by Agrobacterium-mediated transformationof the inbred corn (Zea mays) line A188. Type-I embryogenic callus wastransformed essentially as described in Negrotto et al. (Plant CellReports 19: 798-803, 2000), incorporated herein by reference, using aDNA fragment from plasmid pZM26 (FIG. 1). pZM26 contains a nucleotidesequence comprising tandem expression cassettes. The first expressioncassette is comprised of a MTL promoter sequence (U.S. Pat. No.6,018,099) operably linked to a cry3A055 coding sequence furtheroperably linked to a nopaline synthase 3′ end transcription terminationand polyadenylation sequence. The second expression cassette iscomprised of a maize ubiquitin promoter (ZmUbiInt) (Christensen et al.1992 PMB 18: 675) operably linked to a pmi coding sequence furtheroperably linked to a nopaline synthase 3′ end transcription terminationand polyadenylation sequence.

Immature embryos were excised from 8-12 day old ears and rinsed withfresh medium in preparation for transformation. Embryos were mixed withthe suspension of Agrobacterium cells harboring the transformationvector pZM26, vortexed for 30 seconds, and allowed to incubate for anadditional 5 minutes. Excess Agrobacterium solution was aspirated andembryos were then moved to plates containing a non-selective culturemedium. Embryos were co-cultured with the remaining Agrobacterium at 22°C. for 2-3 days in the dark. Embryos were transferred to culture mediumsupplemented with ticarcillin (100 mg/ml) and silver nitrate (1.6 mg/l)and incubated in the dark for 10 days. Embryos producing embryogeniccallus were transferred to cell culture medium containing mannose.

Regenerated plantlets were tested by TAQMAN® PCR analysis (see Example2) for the presence of both the pmi and cry3A055 genes, as well as forthe absence of the antibiotic resistance spectinomycin (spec) gene.Plants positive for both transgenes, and negative for the spec gene,were transferred to the greenhouse for further propagation. Positiveevents were identified and screened using insect bioassays against cornrootworm. Insecticidal events were characterized for copy number byTAQMAN analysis. MIR604 was chosen for further analysis based on havinga single copy of the transgenes, good protein expression as identifiedby ELISA, and good insecticidal activity against corn rootworm.

The T₀ MIR604 was backcrossed to inbred corn line CG00526, creating theT₁ population. The T₁ plants were self-pollinated to create the T₂generation, and this process was repeated to create a T₃ generation.Progeny testing of the T₃ plants was employed to identify homozygous(converted) families. The MIR604-converted CG00526 inbred was crossed toother elite inbred lines to create hybrids used in further studies.

Example 2 MIR604 Detection by TAQMAN PCR

TAQMAN analysis was essentially carried out as described in Ingham etal. (Biotechniques, 31:132-140, 2001) herein incorporated by reference.Briefly, genomic DNA was isolated from leaves of transgenic andnon-transgenic corn plants using the Puregene® Genomic DNA Extractionkit (Gentra Systems, Minneapolis, Minn.) essentially according to themanufacturer's instruction, except all steps were conducted in 1.2 ml96-well plates. The dried DNA pellet was resuspended in TE buffer (10 MmTris-HCl, pH 8.0, 1 mM EDTA).

TAQMAN PCR reactions were carried out in 96-well plates. For theendogenous corn gene control, primers and probes were designed specificto the Zea mays alcohol dehydrogenase (adh) gene (Genbank accession no.AF044295). It will be recognized by the skilled person that other corngenes can be used as endogenous controls. Reactions were multiplexed tosimultaneously amplify cry3A055 and adh or pmi and adh. For each sample,a master mixture was generated by combining 20 μL extracted genomic DNAwith 35 μL 2× TAQMAN Universal PCR Master Mix (Applied Biosystems)supplemented with primers to a final concentration of 900 nM each,probes to a final concentration of 100 nM each, and water to a 70 μLfinal volume. This mixture was distributed into three replicates of 20μL each in 96-well amplification plates and sealed with optically clearheat seal film (Marsh Bio Products). PCR was run in the ABI Prism 7700instrument using the following amplification parameters: 2 min at 50° C.and 10 min at 95° C., followed by 35 cycles of 15 s at 95° C. and 1 minat 60° C.

Results of the TAQMAN analysis demonstrated that event MIR604 had onecopy of the cry3A055 gene and one copy of the pmi gene.

Examples of suitable primer/probe sequence combinations which were usedare:

Primer Name Primer Sequence SEQ ID NO: Cry3A055-forward5′-TACGAGAGGTGGGT SEQ ID NO: 47 GAACTTGA-3′ Cry3A055-reverse5′-CGATCAGGTCCAGC SEQ ID NO: 48 ACGG-3′ Cry3A055-probe 5′-CCGCTACCGCCGCGSEQ ID NO: 49 AGATGA-3′ (5′ label = FAM, 3′ label = TAMRA) PMI-forward5′-CCGGGTGAATCAGC SEQ ID NO: 50 GTTT-3′ PMI-reverse 5′-GCCGTGGCCTTTGASEQ ID NO: 51 CAGT-3′ PMI-probe 5′-TGCCGCCAACGAAT SEQ ID NO: 52CACCGG-3′ (5′ label = FAM, 3′ label = TAMRA) ZmADH-267 forward5′-GAACGTGTGTTGGG SEQ ID NO: 53 TTTGCAT-3′ ZmADH-337 reverse5′-TCCAGCAATCCTTG SEQ ID NO: 54 CACCTT-3′ ZmADH-316 probe5′-TGCAGCCTAACCAT SEQ ID NO: 55 GCGCAGGGTA-3′ (5′ label = TET, 3′ label= TAMRA)

Example 3 MIR604 Detection by Southern Blot

Genomic DNA used for southern analysis was isolated from pooled leaftissue of ten plants representing the backcross six (BC6) generation ofMIR604 using essentially the method of Thomas et al. (Theor. Appl.Genet. 86:173-180, 1993), incorporated herein by reference. All plantsused for DNA isolation were individually analyzed using TAQMAN PCR (asdescribed in Example 2) to confirm the presence of a single copy of thecry3A055 gene and the pmi gene. For the negative segregant controls, DNAwas isolated from pooled leaf tissue of five plants representing the BC4generation of event MIR604. These negative segregant plants wereindividually analyzed using TAQMAN PCR and the assays were negative forthe presence of the cry3A055 gene and the pmi gene, but were, asexpected, positive for the assay internal control, the endogenous maizeadh gene.

Southern analysis was carried out using conventional molecular biologytechniques. Genomic DNA (7.5 μg) was digested with KpnI restrictionenzyme, which has a single recognition site within the MIR604 T-DNAinsert from plasmid pZM26 (FIG. 1). This approach allows fordetermination of the number of copies of the elements, corresponding tothe specific probe used for each Southern, which have been incorporatedinto MIR604. This results in one hybridization band per copy of theelement present in MIR604. Following agarose gel electrophoresis andalkaline transfer to a Nytran® membrane, hybridizations were carried outusing element-specific full-length PCR-generated probes. The probe usedin the cry3A055 and pmi Southern blots comprise the nucleotide sequencesset forth in SEQ ID NO: 58 and SEQ ID NO: 61, respectively. The probeswere labeled with ³²P via random priming using the Rediprime™ II system(Amersham Biosciences, Cat. No. RPN1633).

The following high stringency hybridization conditions were used: 1-2million cpm/ml are added to PerfectHyb (Sigma) supplemented with 100μg/ml Calf Thymus DNA (Invitrogen) pre-warmed to 65° C.Pre-hybridization takes place in the same solution as above, at the sametemp overnight or for at least one hour. Hybridization was carried outat 65° C. for 3 hours followed by washing 2× in 2×SSC, 0.1% SDS for 20minutes at 65° C. and 2× in 0.1×SSC, 0.1% SDS for 20 minutes at 65° C.

Included on each Southern were three control samples: (1) DNA from anegative (non-transformed) segregant used to identify any endogenous Zeamays sequences that may cross-hybridize with the element-specific probe;(2) DNA from a negative segregant into which is introduced an amount ofKpnI-digested pZM26 that is equal to one copy number based on probelength, to demonstrate the sensitivity of the experiment in detecting asingle gene copy within the Zea mays genome; and (3) KpnI-digested pZM26plasmid that is equal to one copy number based on probe length, as apositive control for hybridization as well as to demonstrate thesensitivity of the experiment.

The hybridization data provide confirmatory evidence to support theTAQMAN PCR analysis that MIR604 contains a single copy of the cry3A055and pmi genes, and that MIR604 does not contain any of the vectorbackbone sequences present in pZM26. As expected for both the cry3A055and pmi probes, the KpnI digest resulted in a single hybridization bandof the correct size, demonstrating that a single copy of each gene ispresent in the MIR604 event. Additionally, for the backbone probe lackof hybridization demonstrates the absence of any pZM26 vector backbonesequences being incorporated into MIR604 during the transformationprocess.

Example 4 T-DNA Insert Sequencing

The nucleotide sequence of the entire transgene DNA insert present inevent MIR604 was determined to demonstrate overall integrity of theinsert, contiguousness of the functional elements and to detect anyindividual basepair changes. The MIR604 insert was PCR amplified fromDNA derived from the BC5 generation as two individual overlappingfragments. Each fragment was amplified using one polynucleotide primerhomologous to plant genomic sequences flanking the MIR604 insert and onepolynucleotide primer homologous to the cry3A055 gene. To generate the5′ fragment, a first polynucleotide primer homologous to the 5′ flankingsequence, 5′S1 (SEQ ID NO: 15), was combined with a secondpolynucleotide primer homologous to the inserted DNA within the cry3A055gene, 5′AS1 (SEQ ID NO: 28). To generate the 3′ fragment, a firstpolynucleotide primer homologous to the 3′ flanking sequence, 9268AS(SEQ ID NO: 45), was combined with a second polynucleotide primerhomologous to the inserted DNA within the cry3A055 gene, 5161S (SEQ IDNO: 27).

PCR amplification was carried out using the Expand High Fidelity PCRsystem (Roche, Cat. No. 1732650) and the following amplificationparameters: 2 min at 94° C. for 1 cycle, followed by 10 cycles of 15 sat 94° C., 30s at 55-65° C. and 5 min at 68° C., followed by 20 cyclesof 15s 94° C., 30s at 55-65° C., and 5 min+5s/cyc of 72° C., followed by1 cycle of 7 min at 72° C.

The amplicon resulting from the PCR amplification using SEQ ID NO: 15and SEQ ID NO: 28 comprised the 5′ junction sequence (SEQ ID NO: 1). Theamplicon resulting from the PCR amplification using SEQ ID NO: 45 andSEQ ID NO: 27 comprised the 3′ junction sequence (SEQ ID NO: 2). Eachsequencing fragment was individually cloned into the pCR®-XL-TOPO vector(Invitrogen, Cat. No. K4700-20) and three separate clones for eachfragment were identified and sequenced. Sequencing was carried out usingthe ABI3730XL analyzer using ABI BigDye® 1.1 or Big Dye 3.1 dGTP (for GCrich templates) chemistry. The sequence analysis was done using thePhred, Phrap, and Consed package from the University of Washington andwas carried out to an error rate of less than 1 in 10,000 bases (Ewingand Green, 1998). The final consensus sequence was determined bycombining the sequence data from the six individual clones (three foreach sequencing fragment) to generate one consensus sequence of theMIR604 insert. To further validate any individual basepair discrepanciesbetween the MIR604 insert and the pZM26 plasmid, small (approximately300-500 bp) PCR products specific to any regions where a basepairdiscrepancy was seen in the initial consensus sequence were amplifiedusing the same methodology above. For all putative basepairdiscrepancies in the MIR604 insert, direct PCR product sequencingresulted in single clear peaks at all basepairs in question, indicatingthese discrepancies are likely present in the MIR604 insert. Alignmentwas performed using the ClustalW program with the following parameters:scoring matrix blosum55, gap opening penalty 15, gap extension penalty6.66 (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680).

The consensus sequence data for the MIR604 T-DNA insert demonstratesthat the overall integrity of the insert and contiguousness of thefunctional elements within the insert as intended in pZM26 have beenmaintained. Sequence analysis revealed that some truncation occurred atthe right border (RB) (SEQ ID NO: 57) and left border (LB) (SEQ ID NO:62) ends of the T-DNA insert during the transformation process thatresulted in event MIR604. The RB portion of the T-DNA insert wastruncated by 44 bp and the LB end of the T-DNA insert was truncated by43 bp. These deletions have no effect on the efficacy of the T-DNAinsert and this phenomenon has been previously observed in Agrobacteriumtransformation (Tinland & Hohn, 1995. Genetic Engineering, 17: 209-229).Additionally, three base pair changes were noted in the MIR604 T-DNAinsert. One discrepancy occurred within the MTL promoter, a regulatoryregion that does not encode a protein. The remaining two discrepanciesoccurred within the pmi coding sequence and did result in two amino acidchanges; valine at position 61 has been substituted by alanine (V61A)and glutamine at position 210 has been substituted by histidine (Q210H).Alanine and valine are both aliphatic amino acids resulting in aconservative substitution. Replacement of glutamine with histidineresults in the substitution of an acidic residue for a basic residue.

Example 5 Analysis of Flanking DNA Sequence

Corn genome DNA sequence flanking the heterologous DNA inserted into thecorn plant genome of event MIR604 was obtained using OmniPlex™Technology essentially as described in Kamberov et al (Proceedings ofSPIE, Tools for Molecular Analysis and High-Throughput Screening,4626:1-12, 2002), incorporated herein by reference.

The 5′ and 3′ flanking sequences and junction sequences were confirmedusing standard PCR procedures. The 5′ flanking and junction sequenceswere confirmed using a first polynucleotide primer set forth in SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12 or SEQ ID NO: 13 combined with a second polynucleotide primer setforth in SEQ ID NO: 16 or SEQ ID NO: 17. The 3′ flanking and junctionsequences were confirmed using a first polynucleotide primer set forthin SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43 or SEQ ID NO: 44 combined with a second polynucleotide primer setforth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQID NO: 35 or SEQ ID NO: 36. It will be recognized by the skilled personthat other primer sequences can be used to confirm the flanking andjunction sequences.

The MIR604 insert was found to be flanked on the right boder (5′flanking sequence) by the corn genomic sequence shown in SEQ ID NO: 5and flanked on the left border (3′ flanking sequence) by the corngenomic sequence shown in SEQ ID NO: 6. The 5′ junction sequence is setforth in SEQ ID NO: 1. The 3′ junction sequence is set forth in SEQ IDNO: 2.

Example 6 Detection of MIR604 Protein via ELISA

To characterize the range of expression of Cry3A055 (the activeinsecticidal principle) and phosphomannose isomerase (PMI) (theselectable marker) proteins in MIR604 plants, the concentrations ofCry3A055 protein and PMI were determined by ELISA in several planttissues and whole plants at four growth stages (whorl, anthesis, seedmaturity and senescence) in two hybrids (MIR604-B and MIR604-C) and oneinbred (MIR604-A). The hybrids were hemizygous for the transgenes inevent MIR604, whereas the inbred was homozygous for the transgenes.

Whole plants and individual parts (except pollen) were reduced to a finepowder by processing using either a coffee grinder, blender, Grindomix™grinder (Brinkmann Instruments; Westbury, N.Y., USA), mortar with apestle or mill, or a combination of these devices. All processing wasdone in the presence of either dry ice or liquid nitrogen. Samples weremixed well to ensure homogeneity. The entire plant tissue sample, or arepresentative sub-sample, was retained for analysis, allowingsufficient sample size for archival storage of reserve plant tissuesamples. The percent dry weight of each sample was determined and theprocessed samples were stored at ca. −80° C. until lyophilization.

Fresh tissue (except pollen and silage) and whole-plant samples wereextracted. For each sample analyzed, a 1.0 g aliquot of the powderedfresh material was weighed into a 15-ml polypropylene tube, suspended in3 ml extraction buffer [50 mM CAPS, 0.1 M NaCl, 2 mM EDTA, 1 mMdithiothreitol, 1 mM 4-(1-aminoethyl)benzenesulfonyl fluoride HCl, 1 mMleupeptin, pH 10], and extracted using an Autogizer® homogenizer(Tomtek; Hamden, Conn., USA). After centrifugation for 15 min at10,000×g at 4° C., the supernatant was used for Cry3A055 and PMIanalysis by ELISA. After treatment with iodoacetamide as described byHill and Straka (1988), total protein in the extracts was quantitatedusing the BCA™ Protein Assay Reagent (Pierce; Rockford, Ill., USA).

Pollen extracts were prepared by suspending pollen 1:30 (w/v) inextraction buffer. After 30 min on ice, the pollen suspensions weredisrupted by three passages through a French pressure cell at ca. 15,000psi, followed by centrifugation at 14,000×g for 5 min at 4° C. Cry3A055and PMI analyses by ELISA were performed on the supernatants asdescribed below. Total protein was quantitated as described above.

Silage extracts were prepared by suspending silage 1:25 (w/v) in 2×extraction buffer. After 30 min on ice, the silage suspensions wereextracted using a Brinkmann Polytron® Homogenizer (Brinkmann; Westbury,N.Y., USA). After centrifugation for 15 min at 10,000×g at 4° C., thesupernatant was used for Cry3A055 and PMI analysis by ELISA. Totalprotein was quantitated as described above.

Cry3A055 Quantification

The extracts prepared as described above were quantitatively analyzedfor Cry3A055 by ELISA (Tijssen, 1985) using immuno-affinity purifiedrabbit anti-Cry3A055 polyclonal antibodies and immuno-affinity purifiedgoat anti-Btt (native Cry3A from Bacillus thuringiensis subsp.tenebrionis) polyclonal antibodies. The lower limit of quantification ofthe double-sandwich ELISA was estimated based on the lowestconcentration of pure reference protein lying on the linear portion ofthe standard curve, the maximum volume of a control extract that couldbe analyzed without background interference, and the correspondingweight of the sample that the aliquot represented.

Quantifiable levels of Cry3A055 protein were detected in allMIR604-derived plant tissues analyzed except pollen. In most cases,results are presented as means of the five replicate tissue samples. Forsilage, one sample was analyzed; therefore, no mean could be calculated.Control sample levels were below the limit of quantification for allstages and tissues.

Across all growth stages, mean Cry3A055 levels measured in leaves, rootsand whole plants ranged from ca. 3-23 μg/g fresh wt. (4-94 μg/g drywt.), ca. 2-14 μg/g fresh wt. (7-62 μg/g dry wt.), and about 0.9-11 μg/gfresh wt. (3-28 μg/g dry wt.), respectively. Mean Cry3A055 levelsmeasured in kernels at seed maturity and senescence ranged from about0.6-1.4 μg/g fresh wt. (0.8-2.0 μg/g dry wt.). Mean Cry3A055 levelsmeasured in silk tissue at anthesis were below the lower limit ofquantification (LOQ), <0.1 μg/g fresh wt. (<1.0 μg/g dry wt.). MeanCry3A055 levels measured in silk tissue at seed maturity ranged fromabout 0.6-1.9 μg/g fresh wt. (1-3 μg/g dry wt.). No Cry3A055 protein wasdetectable in pollen from either inbred MIR604-A or hybrids MIR604-B andMIR604-C [limit of detection (LOD)=0.07 μg/g fresh wt., 0.15 μg/g drywt.].

The levels of Cry3A055 were generally similar between hybrids for eachtissue type at each time point. For the inbred line, Cry3A055 expressionwas generally higher than in the hybrids in leaves, roots and wholeplants at whorl and anthesis stages, and in roots at seed maturity.Cry3A055 levels measured in silage tissues were on average 2.5 μg/gfresh wt. (7.3 μg/g dry wt.) over 15, 29 and 75 days. By comparison, thelevel of Cry3A055 measured in the chopped plant material prior toensiling (Day 0 pre-silage) was about 8 μg/g fresh wt. (20 μg/g drywt.).

PMI Quantification

The extracts prepared as described above were quantitatively analyzedfor PMI by ELISA (Tjissen, 1985) using Protein A-purified polyclonalrabbit and immunoaffinity-purified polyclonal goat antibodies specificfor PMI. The lower limit of quantification of the double-sandwich ELISAwas estimated based on the lowest concentration of pure referenceprotein lying on the linear portion of the standard curve, the maximumvolume of a control extract that could be analyzed without backgroundinterference, and the corresponding weight of the sample that thealiquot represented.

PMI protein was detected in most of the MIR604-derived plant tissuesanalyzed, albeit at low levels. In most cases, results are presented asmeans of the five replicate tissue samples. For silage, one replicatewas analyzed; therefore, no mean could be calculated. Control samplelevels were below the limit of quantification for all stages andtissues.

Across all plant stages, mean PMI levels measured in leaves, roots andwhole plants ranged from not detectable (ND) to ca. 0.4 μg/g fresh wt.(ND-2.1 μg/g dry wt.), below the LOQ (<0.03 μg/g fresh wt.) to about 0.2μg/g fresh wt. (<0.1-1.0 μg/g dry wt.), and below the LOQ (<0.02 μg/gfresh wt.) to about 0.3 μg/g fresh wt. (<0.04-2 μg/g dry wt.),respectively. Mean PMI levels measured in kernels at seed maturity andsenescence ranged from below the LOQ (<0.06 μg/g fresh wt.) to about 0.4μg/g fresh wt. (<0.07-0.5 μg/g dry wt.). Mean PMI levels measured insilk tissue at anthesis and seed maturity ranged from below the LOQ(<0.1 μg/g fresh wt.) to about 0.8 μg/g fresh wt. (<0.2-6.8 μg/g drywt.). PMI in pollen ranged from about 1.9-2.6 μg/g fresh wt. (3.9-5.2μg/g dry wt.).

The levels of PMI were generally similar among the inbred and hybridgenotypes for each tissue type at each time point. PMI was notdetectable in silage at all three sampling times (day 15, 29 and 75),whereas the level measured in the chopped plant material prior toensiling (Day 0 pre-silage) was about 0.3 μg/g fresh wt. (0.7 μg/g drywt.).

Estimated Total Cry3A055 Protein Levels Per Acre and Per Hectare

For the inbred line (MIR604-A) and both hybrids (MIR604-B and MIR604-C),the plants reached their highest biomass at seed maturity. The plantsalso reached their highest estimated mean Cry3A055 levels on a per-acre(and per-hectare) basis at seed maturity and were estimated to containabout 78, 141 and 240 g Cry3A055/acre (193, 348 and 592 g/hectare) forMIR604-A, MIR604-B and MIR604-C, respectively. Over the growing seasonand across genotypes, estimates of Cry3A055 in MIR604-derived plantsranged from mean levels of about 8 g Cry3A055/acre (21 gCry3A055/hectare) at senescence stage to about 240 g Cry3A055/acre (592g Cry3A055/hectare) at seed maturity, assuming a planting density of26,500 plants per acre (65,500 plants/hectare).

Example 7 Field Efficacy of MIR604 Western and Northern Corn Rootworm

MIR604 plants were tested for efficacy against western and northern cornrootworm at 12 locations in the United States. MIR604 was tested withand without the addition of the insecticidal seed treatment Crusier®.Control groups consisted of seed treated with two different rates ofCruiser® and an untreated check. Treatments consisted of fourreplications of two 17.5-20 foot rows spaced 30″ on center designed in arandomized complete block. Ten plants per treatment were chosen atrandom and evaluated for efficacy using a 0-3 scale wherein 0=No feedingdamage (lowest rating that can be given); 1=One node (circle of roots),or the equivalent of an entire node, eaten back within approximately twoinches of the stalk (soil line on the 7^(th) node); 2=Two complete nodeseaten; 3=Three or more nodes eaten (highest rating that can be given).Damage in between complete nodes eaten was noted as the percentage ofthe node missing, i.e. 1.50=1½ nodes eaten, o.25=¼ of one node eaten.

Results, shown in Table 1, demonstrate that the roots of two siblinglines of MIR604, 3-11 and 3-12, sustained significantly less feedingdamage than roots from either Cruiser® treatment or the untreatedcontrol roots. MIR04-3-11 and MIR604-3-12 had root damage ratings of0.44 and 0.42, respectively, compared to the 0.25 and 1.25 mgA/SeedCruiser® treatments, which had damage ratings of 1.6 and 0.9,respectively, and the control line with a damage rating of 2.14. Therewas a trend toward lower root damage ratings in the MIR604 plants whoseseed was treated with Cruiser®, suggesting that Crusier® augmented theCry3A055 protein or that there was a possible synergy between Crusier®and Cry3A055. This was particularly evident in the 1.0 and 1.25mgA/MIR604 seed treatments with root damage ratings of 0.33 and 0.29,respectively.

TABLE 1 Efficacy of MIR604 with and without Crusier ® seed treatment.Cruiser ® Treatment Root Damage Rating (0-3 Corn Line (mgA/Seed) CRWScale) MIR604-3-11 0 0.44 MIR604-3-12 0 0.42 MIR604 0.25 0.43 MIR6040.50 0.39 MIR604 1.0 0.33 MIR604 1.25 0.29 Control Hybrid 0.25 1.60Control Hybrid 1.25 0.99 Control Hybrid 0 2.14

MIR604 efficacy was compared with commercial granular insecticidestandards applied in-furrow. The experimental design was as describedabove. Results in Table 2 demonstrate that the efficacy of MIR604 wascomparable to the commercial standards in protecting plants against cornrootworm feeding damage.

TABLE 2 Comparison of efficacy of MIR604 with commercial insecticidesapplied in-furrow. Treatment Root Damage Rating (0-3 CRW Scale) MIR6040.43 Force ® 3G 0.44 Aztec ® 6.7G 0.32 Lorsban ® 15 G 0.75 UntreatedCheck 2.14

Mexican Corn Rootworm

MIR604 plants were evaluated for resistance to the Mexican corn rootwormat two locations in Texas. Experimental design was essentially the sameas described above.

Results shown in Table 3 demonstrate that both MIR604 siblings sustainedless feeding damage than untreated checks. There was a positive responsefor control of Mexican corn rootworm when Cruiser was added to theMIR604 seed. A clear rate response was evident. Results shown in Table 4demonstrate that the efficacy of MIR604 was comparable to the commercialstandards in protecting plants against Mexican corn rootworm feedingdamage.

TABLE 3 Efficacy of MIR604 with and without Cruiser seed treatmentagainst Mexican corn rootworm. Root Damage Rating Treatment Cruiser ®Rate (mgA/Seed) (0-3 CRW Scale) MIR604-3-11 0 1.14 0.125 0.19 0.25 0.180.50 0.09 1.25 0.02 MIR604-3-12 0 0.68 0.125 0.46 0.25 0.18 0.50 0.211.25 0.04 Control Hybrid 0.125 1.59 1.25 0.71 0 2.76

TABLE 4 Efficacy of MIR604 compared with commercial insecticides appliedin-furrow against Mexican corn rootworm. Treatment Root Damage Rating(0-3 CRW Scale) MIR604 0.68 Force ® 3G 0.66 Aztec ® 6.7G 0.88 Lorsban ®15 G 0.81 Untreated Check 2.76

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the present invention.

1. An isolated nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, or the complements thereof.
 2. Thenucleic acid molecule of claim 1, wherein the molecule is comprised in acorn plant, cell, tissue, or seed.
 3. The nucleic acid molecule of claim2, wherein the molecule is comprised in a progeny plant of the cornplant.
 4. The nucleic acid molecule of claim 1, wherein the molecule iscomprised in a biological sample derived from a corn plant, cell,tissue, or seed.
 5. The nucleic acid molecule of claim 4, wherein thebiological sample is selected from the group consisting of corn flour,corn meal, corn starch, and cereals manufactured in whole or in part tocontain corn by-products.
 6. The nucleic acid molecule of claim 1,wherein the molecule is comprised in an extract derived from a sample ofa corn plant, cell, tissue, or seed.
 7. The nucleic acid molecule ofclaim 6, wherein the sample is selected from the group consisting ofcorn flour, corn meal, corn starch, and cereals manufactured in whole orin part to contain corn by-products.
 8. An amplicon comprising thenucleic acid molecule of claim 1.